1. Field of the Invention
The present invention generally relates to antifungal and antibacterial compositions and methods employing such compositions to deter or prevent fungal growth in stored coatings and on susceptible surfaces. More particularly, the present invention relates to such compositions containing antifungal and antibacterial peptides, polypeptides or proteins and to methods of making and using such compositions.
2. Description of the Related Art
Fungal growth on indoor and outdoor surfaces is a major environmental concern today affecting home, work and recreational environments. Not only can fungus (e.g., mold, mildew) be unsightly on exposed surfaces, it can destroy wood, fiber and other materials if left untreated, causing severe damage to buildings and other structures and equipment. Over the past few years it has become increasingly apparent that exposure to certain fungi or their spores can seriously impact the health of humans, pets and other animals. Although fungi are certainly not the only factors that detrimentally affect indoor air quality, in many instances they have been identified as a primary contributor to indoor air quality problems. In fact, the term “sick building syndrome” was recently coined to describe buildings in which various physical, chemical and biological factors, including growing fungi and/or their spores, have severely compromised the air quality leading to discomfort or illness of the occupants. Concerns such as allergies, asthma, infections, and the long-term repercussions of mold toxins are just a few of the many real health effects associated with mold contamination of indoor and outdoor environments.
Fungi (including true fungi, molds and mildews) are eukaryotic organisms that have cell walls, similar to plants, but do not contain chlorophyll. There are between 100,000-200,000 species of fungi, mold and mildew, depending on which classification methods are used. Of particular concern are the pathogenic fungi, which can cause significant harm to individuals who are exposed to them. About 300 species are presently known to be pathogenic for man, but it is thought that there are many other as yet unrecognized fungal pathogens. The field of medical mycology has emerged as a result of the growing number of fungal-related illnesses and deaths.
Fungi grow as saprophytes, i.e., in a suitable moist environment they are able to decompose organic matter to obtain the nourishment needed for growth. Building and decorative materials such as wood, paper-coated wallboard, wallpaper, fabrics, carpet and leather can provide the necessary organic matter. Today, an especially problematic fungal genus sometimes found in buildings that have excess indoor moisture is Stachybotrys. Stachybotrys chartarum, commonly found in nature growing on cellulose-rich plant materials, has often been found in water-damaged building materials, such as ceiling tiles, wallpaper, wallboard and cellulose resin wallboard (fiberboard). Depending on the particular conditions of temperature, pH and humidity in which the mold is growing, Stachybotrys may produce mycotoxins, compounds that have toxic properties.
Other common fungi that can grow in residential and commercial buildings are Aspergillus species (sp.), Penicillium sp., Fusarium sp., Alternaria dianthicola, Aureobasidium pullulans (aka Pullularia pullulans), Phoma pigmentivora and Cladosporium sp. The moist indoor environment which promotes growth of these fungi can arise from water damage, excessive humidity, water leaks, condensation, water infiltration, or flooding, in some cases due to defects in building construction, faulty mechanical system design, and/or operational problems. Even modern homes and commercial buildings are not immune to fungal invasion despite the use of technologically advanced building materials and more energy efficient construction and operation than in buildings of the past. Modern homes tend to be less well ventilated, and although the use of air conditioning reduces humidity making it harder for mold to grow, today's central air conditioning systems can also facilitate the spread of mold spores throughout a home. Increased use of paper products in homes and commercial buildings today further encourages mold growth. Heavy contamination of indoor or outdoor surfaces by dirt and/or oil can also provide a food source for a fungus. Vulnerable structures and materials that are difficult to access for cleaning, or for which cleaning is neglected, are particularly vulnerable to attack by fungi. Fungi are also known to contaminate stored paints, fuels, and many other industrial products.
Fungal colonies typically take on filamentous form, having long filament-like cells called hyphae. Under the right environmental conditions, hyphae grow into an intertwining network called the mycelium. A mycelium can be visible to the naked eye, appearing as unsightly fuzzy green, bluish-gray or black spots, for example. When conditions for growth are less favorable, many varieties of fungi can respond by forming spores on specialized hyphal cells. Spores are the primary means for dispersal and survival of fungi, and can remain dormant for months or even years—even withstanding extremely adverse conditions, to germinate and flourish again when environmental variables such as light, oxygen levels, temperature, and nutrient availability again become favorable. Thick-walled spores are substantially more resistant to common disinfective agents than are the thinner-walled vegetative fungal cells. According to the U.S. Environmental Protection Agency, there is no practical way to eliminate all mold and mold spores in the indoor environment.
As mentioned above, paints and paint films or coatings are known to be vulnerable to mold contamination due to the presence of common organic components that act as cellulosic thickeners, surfactants and defoamers, and which can also serve as a source of food for fungus cells. Some of these components are casein, acrylic, polyvinyl and other carbon polymers. For example, latex is a water-dispersed binder comprising a carbon polymer. Inside the paint can, certain fungi (e.g., yeasts) can convert enough carbon-containing food sources to CO.sub.2 to swell or even explode the can. Fungi can also discolor and reduce the viscosity of the paint, and produce foul odors. Both in-can preservation of paints and protection of the end use paint films, and the surfaces they cover, from mold, mildew and yeasts is necessary. To combat fungi, a variety of coating materials have been formulated which include organic or inorganic chemicals to discourage or prevent the growth of mildew on the paint film. Ideally, these chemical fungicides or mildewcides slowly leach out of the paint to the surface, and maintain their inhibitory properties for the life of the paint film, causing little or no harm to the environment. In practice, however, the antifungal properties of most coating compositions in use today persist for variable lengths of time, depending on the amount of exposure to the elements, abrasion and erosion.
Most antifungal chemicals are non-specific as to the organism affected and can be detrimental to the environment, including toxicity to plant and animal life. It is more difficult to identify fungus-specific agents than it is to discover bacteria specific-agents because fungal cells share many similarities with the cells of higher organisms, whereas bacterial cells are distinctly different. For this reason, fungicides tend to be more toxic to humans and animals than are bactericides. U.S. Pat. No. 5,882,731 (Owens) describes a number of common and proprietary chemical mildewcide-containing products that have been investigated as additives for water-based latex mixtures. Some known antifungal agents that have been used in the coatings industry are: copper (II) 8-quinolinolate (CAS No. 10380-28-6); zinc oxide (CAS No. 1314-13-2); zinc-dimethyl dithiocarbamate (CAS No. 137-30-4); 2-mercaptobenzothiazole, zinc salt (CAS No. 155-04-4); barium metaborate (CAS No. 13701-59-2); tributyl tin benzoate (CAS No. 4342-36-3); bis tributyl tin salicylate (CAS No. 22330-14-9), tributyl tin oxide (CAS No. 56-35-9); parabens: ethyl parahydroxybenzoate (CAS No. 120-47-8), propyl parahydroxybenzoate (CAS No. 94-13-3) methyl parahydroxybenzoate (CAS No. 99-76-3) and butyl parahydroxybenzoate (CAS No. 94-26-8); methylenebis(thiocyanate) (CAS No. 6317-18-6); 1,2-benzisothiazoline-3-one (CAS No. 2634-33-5); 2-mercaptobenzo-thiazole (CAS No. 149-30-4); 5-chloro-2-methyl-3(2H)-isothiazolone (CAS No. 57373-19-0); 2-methyl-3(2H)-isothiazolone (CAS No. 57373-20-3); zinc 2-pyridinethiol-N-oxide (CAS No. 13463-41-7); tetra-hydro-3,5-di-methyl-2H-1,3,5-thiadiazine-2-thione (CAS No. 533-74-4); N-trichloromethyl-thio-4-cyclohexene-1,2-dicarboximide (CAS No. 133-06-2); 2-n-octyl-4-isothiazoline-3-one (CAS No. 26530-20-1); 2,4,5,6-tetrachloro-isophthalonitrile (CAS No. 1897-45-6); 3-iodo-2-propynyl butylcarbamate (CAS No. 55406-53-6); diiodomethyl-p-tolylsulfone (CAS No. 20018-09-1); N-(trichloromethyl-thio)phthalimide (CAS No. 133-07-3); potassium N-hydroxy-methyl-N-methyl-dithiocarbamate (CAS No. 51026-28-9); sodium 2-pyridinethiol-1-oxide (CAS No. 15922-78-8); 2-(thiocyanomethylthio)benzothiazole (CAS No. 21564-17-0); 2-4(-thiazolyl)benzimidazole (CAS No. 148-79-8). See V. M. King, “Bactericides, Fungicides, and Algicides,” Ch. 29, pp. 261-267; and D. L. Campbell, “Biological Deterioration of Paint Films,” Ch. 54, pp. 654-661; both in PAINT AND COATING TESTING MANUAL, 14.sup.th ed. of the Gardner-Sward Handbook, J. V. Koleske, Editor (1995), American Society for Testing and Materials, Ann Arbor, Mich. Currently, the Pesticide Action Network North America (PAN) lists in its Internet chemical database, the above-mentioned chemicals plus more than 700 additional chemicals designated as pesticides having antifungal properties in soil and wood.
The mode of action of some of the metal-based antifungal agents is thought to be chelation of metals that are necessary to growth of the organisms. Some of the nitrogen- and/or sulfur-containing antifungal agents are thought to act by uncoupling oxidative phosphorylation in the fungal cells, or inhibiting oxidation of glucose. The paraben compounds (aka hydroxybenzoate) are thought to affect membrane activity and integrity.
Due to environmental and safety concerns, there is increasing pressure today on the coatings industry to eliminate some of the more effective but more toxic chemical preservatives from paints and other coating compositions. Yet at the same time, consumers wish to avoid purchasing spoiled or poorly performing products. Thus, there is a great need in the industry today for safe and effective alternatives to conventional antifungal agents.
Various naturally occurring biological products that are said to possess antifungal activity are described in the background discussion of U.S. Pat. Nos. 6,020,312; 5,602,097; and 5,885,782 [each incorporated in their entirety by reference herein]. In many cases, the active component of those natural antifungal agents has not been identified nor completely characterized. Since most of the known naturally occurring antifungal agents are poorly characterized at best, the persistence and toxicity of such compounds in the environment is also unknown. Furthermore, the fact that many of those compounds are produced by microbes in the environment suggests that they may have a limited spectrum of antifungal activity. A drawback of most of the antifungal agents in use today is that they are as toxic to higher organisms as they are to the target fungi. The more target-specific antifungal agents tend to be very rare and/or costly.
Recently developed methods permit the preparation of synthetic peptide combinational libraries (“SPCLs”) that are composed of equimolar mixtures of free peptides that can be used with in vitro methods to determine bioactivity (Furka, A., et al. Int. J. Pept. Protein Res. 37:487 (1991), Houghten, R. A., et al. Nature 354:84 (1991), Houghten, R. A., et al. BioTechniques 13:412 (1992). Libraries can consist of D- or L-amino acid stereoisomers or combinations of L- and D- and/or non-naturally-occurring amino acids. Other methods for synthesizing peptides of defined sequence are also known. Similarly, large-scale preparative methods are known. Certain recombinant methods for producing peptides have also been described, e.g., U.S. Pat. No. 4,935,351. While U.S. Pat. Nos. 6,020,312; 5,602,097; and 5,885,782 describe agricultural uses for certain synthetic antifungal peptides, none of those or any other peptidic agents have been previously investigated as additives for use in the paints and coatings industry.
Although significant advancement has been made in identifying various chemical agents and natural and synthetic peptides or proteins that demonstrate antifungal activity for certain uses (e.g., medical treatment or agricultural use), there is no indication that any such biomolecule could be used successfully in paints or other coating materials for protecting or treating non-living objects. Antifungal or fungus-resistant paints and other coating compositions are needed which do not suffer from the same limitations as conventional surface coating materials containing existing fungicides and antifungal agents. Ideally, an antifungal paint will contain fungus-specific fungus deterring/inhibiting/killing agents that are stable in paints and other coating mixtures during storage, persist in the resulting coat or film that is spread out over a surface in need of protection from fungal infestation, and which are safer to the environment. Better antifungal materials would be especially welcomed by original equipment manufacturers (OEMs), and by the architectural, marine and industrial maintenance industries. In particular, antifungal and antibacterial additives to paints and coatings that work alone or synergistically with existing antifungal agents would be desirable.